The present invention relates to the field of the diagnosis of neurological diseases. The present invention provides a new method for the differential diagnosis of Alzheimer""s disease versus other neurological diseases. More particular, the present invention provides a method for the differential diagnosis of Alzheimer""s disease versus dementia with Lewy bodies, versus Parkinson""s disease without dementia, versus multi-system atrophy and/or versus progressive supranuclear palsy.
The use of tau and phospho-tau as neurological markers in the diagnosis of neurological diseases has been postulated (Blennow et al., 1995; Vigo-Pelfrey et al., 1995; Andreasen et al., 1998; Andreasen et al., 1999a; Ishiguro et al., 1999). The microtubule-associated protein tau is a major protein component of paired helical filaments (PHF) and neurofibrillar tangles (NFT), associated with Alzheimer""s disease (Brion et al., 1985; Delacourte and Defossez, 1986; Grundke-Iqbal et al., 1986; Kosik et al., 1986; Wood et al., 1986; Kondo et al., 1988). Tau protein exists in different isoforms, of which 4 to 6 are found in adult brain but only 1 isoform is detected in fetal brain. The diversity of the isoforms is generated from a single gene on human chromosome 17 by alternative mRNA splicing (Himmler, 1989; Goedert et al., 1989; Andreadis et al., 1992). The most striking feature of tau protein, as deduced from molecular cloning, is a stretch of 31 or 32 amino acids, occurring in the carboxy-terminal part of the molecule, which can be repeated either 3 or 4 times. Additional diversity is generated through 29 or 58 amino acid-long insertions in the NH2-terminal part of tau molecules (Goedert et al., 1989). In vivo tau promotes microtubule assembly and stability in the axonal compartment of neurons by interactions involving its microtubule binding domain which is localized in the repeat region of tau (255-381) (Lewis et al., 1988). In normal circumstances adult brain contains 2-3 mole phosphate per mole of tau (Selden and Pollard, 1983; Ksiezak-Reding et al., 1992). Phosphorylation of different sites in normal tau as studied in rat and humans is dependent on the developmental state (Lee et al., 1991; Bramblett et al., 1993; Goedert et al., 1993). Tau variants of 60, 64 and 68 kDa arising as a consequence of phosphorylation have been detected in brain areas showing neurofibrillary tangles (Delacourte et al., 1990; Goedert et al., 1992; Flament et al., 1990, Greenberg and Davies, 1990). These brains contain 6-8 mole phosphate per mole tau (Ksiezak-Reding et al., 1992). In tau isolated from PHF (PHF-tau), phosphorylation occurs at several positions (Iqbal et al., 1989; Lee et al., 1991; Hasegawa et al., 1992; Hanger et al., 1998; Buee et al., 1999).
Alzheimer""s disease (AD) and frontotemporal dementia (FTD) are the most common types of primary degenerative dementia associated with a tau pathology, having a prevalence of respectively 42-75% and 8-10% (Brun, 1993; Gustafson, 1993; Ebly et al., 1994). Filamentous tau pathology i.e. neurofibrillary tangles (NFT), are consistently found in AD (Tomlinson and Corsellis, 1984) but may also be found in FTD (Spillantini and Goedert, 1998). Pathological tau proteins are found both in AD and FTD (Vermersch et al., 1995; Delacourte et al., 1996). Studies on brain tissue, however, have suggested that the tau pathology differs between AD and FTD, possibly being related to the degree of phosphorylation (Delacourte et al., 1996). Other forms of dementia associated with a tau pathology include familial FTD, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and subacute sclerosing panencephalitis. The role of hyperphosphorylation in the pathology of these tauopathies is at present not well understood.
Dementia with Lewy bodies (DLB) is an illness that presents with progressive dementia or psychosis. Parkinsonian signs, which may be absent or mild at the onset, eventually become common and rigidity is usually severe. Lewy bodies are found profusely in the brainstem, basal forebrain, hypothalamic nuclei and neocortex. Dementia with Lewy bodies is characterized by the relative absence of tangles and hyperphosphorylated tau in the brain. Parkinson""s disease (PD) is a type of Lewy Body disease occurring in the middle or late life, with very gradual progression and a prolonged course. It can be considered as an example of neuronal system disease, involving mainly the nigrostriatal dopaminergic system. Dementia with Lewy bodies was recently defined as a special form of dementia requiring differential patient management (Lebert et al., 1998; McKeith et al., 1999). Dementia with Lewy bodies, which is sensitive to neuroleptics, is clinically very difficult to differentiate from Alzheimer""s disease (McKeith et al., 1996; Ballard et al., 1998). Most patients (more than 75%) are neuropathologically defined as Alzheimer""s disease patients while it is estimated that 15 to 25% of the clinically diagnosed Alzheimer""s disease patients have dementia with Lewy bodies (Hooten et al., 1998). As dementia with Lewy bodies is more susceptible to acetylcholinesterase treatment, differentiation of dementia with Lewy bodies from Alzheimer""s disease is essential for optimization of treatment (Levy et al., 1994; Perry et al., 1994; Wilcock et al., 1994).
Cerebrospinal fluid (CSF)-xcex2-amyloid and CSF-tau have been validated to discriminate Alzheimer""s disease from normal aging, depression and Parkinson""s disease (Galasko et al., 1998; Kanai et al., 1998; Hulstaert et al., 1999) and these markers are well suited for the differential diagnosis of these disorders (Andreasen et al., 1999b). More controversial, however, is their role in the discrimination of Alzheimer""s disease from closely related conditions such as dementia with Lewy bodies and from other dementia associated with tau pathology such as frontotemporal dementia, multi-system atrophy (MSA) and/or progressive supranuclear palsy. At present, no accurate methods exist for the differential diagnosis of these neurological diseases.
It is an aim of the present invention to provide a method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from another neurological disease.
It is an aim of the present invention to provide a method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from dementia with Lewy bodies.
It is an aim of the present invention to provide a method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from Parkinson""s disease without dementia.
It is an aim of the present invention to provide a method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from multi-system atrophy.
It is an aim of the present invention to provide a method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from progressive supranuclear palsy.
It is an aim of the present invention to provide an in vitro method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from another neurological disease.
It is an aim of the present invention to provide an in vitro method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from dementia with Lewy bodies.
It is an aim of the present invention to provide an in vitro method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from Parkinson""s disease without dementia.
It is an aim of the present invention to provide an in vitro method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from multi-system atrophy.
It is an aim of the present invention to provide an in vitro method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from progressive supranuclear palsy.
It is an aim of the present invention to provide a diagnostic kit for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from another neurological disease.
It is an aim of the present invention to provide a diagnostic kit for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from dementia with Lewy bodies.
It is an aim of the present invention to provide a diagnostic kit for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from Parkinson""s disease without dementia.
It is an aim of the present invention to provide a diagnostic kit for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from multi-system atrophy.
It is an aim of the present invention to provide a diagnostic kit for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from progressive supranuclear palsy.
It is an aim of the present invention to provide a method to screen or monitor the effect of compounds which prevent dementia with Lewy bodies, Parkinson""s disease without dementia, multi-system atrophy and/or progressive supranuclear palsy or which treat an individual suffering from dementia with Lewy bodies, an individual suffering from Parkinson""s disease without dementia, an individual suffering from multi-system atrophy and/or an individual suffering from progressive supranuclear palsy.
The present invention relates to a method for the differential diagnosis of an individual suffering from Alzheimer""s disease (AD) versus an individual suffering from another neurological disease characterized that phospho-tau is used as a neurological marker. More specifically, the present invention relates to a method as described above comprising the steps of:
determining the level of phospho-tau in said individual;
inference that said individual is suffering from a neurological disease other than Alzheimer""s disease by comparing the obtained level of phospho-tau in said individual with the level of phospho-tau in individuals suffering from Alzheimer""s disease, whereby a decreased level of phospho-tau being an indication that said individual is suffering from a neurological disease other than Alzheimer""s disease.
xe2x80x98Differential diagnosis of an individual suffering from a neurological disease versus an individual suffering from another neurological diseasexe2x80x99 as used in the present invention refers to the discrimination between said first neurological disease and other neurological diseases in this way that a certain neurological disease or a certain cause of neurological disorder in an individual is associated with a certain neurodegenerative condition of said individual. The method of the present invention allows the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from a neurological disease other than Alzheimer""s disease. In a specific embodiment, the present invention allows the differential diagnosis of an individual suffering from Alzheimer""s disease (AD) versus an individual suffering from dementia with Lewy bodies (DLB). In another specific embodiment, the present invention allows the differential diagnosis of an individual suffering from Alzheimer""s disease (AD) versus an individual suffering from Parkinson""s disease (PD) without dementia. In another specific embodiment, the present invention allows the differential diagnosis of an individual suffering from Alzheimer""s disease (AD) versus an individual suffering from multi-system atrophy (MSA). In another specific embodiment, the present invention allows the differential diagnosis of an individual suffering from Alzheimer""s disease (AD) versus an individual suffering from progressive supranuclear palsy (PSP). Alzheimer""s disease, dementia with Lewy bodies, Parkinson""s disease without dementia, multi-system atrophy and progressive supranuclear palsy as well as other neurological diseases have been described in detail by Wilson et al. (1991) and McKeith et al. (1999).
The present invention is based on the finding that the level of phospho-tau in CSF from individuals suffering from dementia with Lewy bodies, from PD without dementia, from MSA or from PSP is significantly decreased compared to the level of phospho-tau in CSF from individuals suffering from Alzheimer""s disease. The indication that the level of phospho-tau differs between said neurological diseases, forms the basis for the development of a diagnostic test for the differential diagnosis of said neurological diseases in an individual. Accordingly, the present invention relates to a method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from dementia with Lewy bodies, versus an individual suffering from Parkinson""s disease without dementia, versus an individual suffering from multi-system atrophy and/or versus an individual suffering from progressive supranuclear palsy, said method characterized that phopho-tau is used as a neurological marker.
The method of the invention thus comprises the step of determining the level of phospho-tau in said individual suspected of suffering from AD, from DLB, from PD without dementia, from MSA or from PSP and comparing it with a previously defined phospho-tau level range characteristic for AD, DLB, PD without dementia, MSA or PSP. A level of phospho-tau falling within the previously defined phospho-tau level range for Alzheimer""s disease is an indication that said individual is suffering from AD. A level of phospho-tau falling within the previously defined phospho-tau level range for DLB is an indication that said individual is suffering from DLB. A level of phospho-tau falling within the previously defined phospho-tau level range for PD without dementia is an indication that said individual is suffering from PD without dementia. A level of phospho-tau falling within the previously defined phospho-tau level range for MSA is an indication that said individual is suffering from MSA. A level of phospho-tau falling within the previously defined phospho-tau level range for PSP is an indication that said individual is suffering from PSP.
Accordingly, the present invention relates to a method for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from dementia with Lewy bodies, versus an individual suffering from Parkinson""s disease without dementia, versus an individual suffering from multi-system atrophy and/or versus an individual suffering from progressive supranuclear palsy, comprising the steps of:
determining the level of phospho-tau in said individual;
inference that said individual is suffering from DLB, from PD without dementia, from MSA and/or from PSP by comparing the obtained level of phospho-tau in said individual with the level of phospho-tau in individuals suffering from AD, whereby a decreased level of phospho-tau being an indication that said individual is suffering from DLB, from PD without dementia, from MSA and/or from PSP.
The level of phospho-tau can be detected in vitro as well as in vivo. The method for the in vitro detection of the level of phospho-tau in an individual comprises the steps of obtaining a sample from said individual, determining the level of phospho-tau in said sample and comparing it with a previously defined phospho-tau level range for said neurological disease.
The term xe2x80x98samplexe2x80x99 refers to any source of biological material, for instance body fluids, brain extract, peripheral blood or any other sample comprising phospho-tau protein. In a preferred embodiment, the level of phospho-tau is determined in vitro by analysis of the level of phospho-tau in a body fluid sample of the patient. The term xe2x80x98body fluidxe2x80x99 refers to all fluids that are present in the human body including but not limited to blood, lymph, urine and cerebrospinal fluid (CSF) comprising phospo-tau protein. The blood sample may include a plasma sample or a serum sample.
In a preferred embodiment of the present invention the level of phospho-tau is determined in a cerebrospinal fluid sample taken from the patient. In accordance, the present invention relates to a method as described above, comprising the steps of:
obtaining a cerebrospinal fluid sample from said individual;
determining the level of phospho-tau in said cerebrospinal fluid sample;
inference that said individual is suffering from a neurological disease other than AD such as DLB, PD without dementia, MSA and/or PSP by comparing the obtained level of phospho-tau in said cerebrospinal fluid sample with the level of phospho-tau in a cerebrospinal fluid sample from individuals suffering from AD, whereby a decreased level of phospho-tau being an indication that said individual is suffering from another neurological disease such as DLB, PD without dementia, MSA and/or PSP.
The level of phospho-tau as measured in the CSF of the individual suspected to suffer from DLB, from PD without dementia, from MSA and/or from PSP is compared with the level of phospho-tau in the CSF of an individual suffering from AD. A decreased level of CSF-phospho-tau is interpreted as an indication of the individual suffering from DLB, from PD without dementia, from MSA and/or from PSP.
Phospho-tau include all forms of tau that have a phosphorylation on any position of the tau protein sequence, but more specifically it refers to phosphorylations on amino acid positions that are not phosphorylated in human normal tau isolated from adult individuals not suffering from any neurological disorder.
xe2x80x98A decreased level of phospho-tauxe2x80x99 means that the level of phospho-tau measured in the patient is lower than the level of phospho-tau measured in patients suffering form AD. Phospho-tau can be quantified by any method known in the art, including but not limited to the use of antibodies. In a preferred embodiment, phospho-tau is quantified by an immunoassay comprising at least the following steps:
obtaining a sample from the patient;
bringing said sample into contact with a monoclonal antibody specifically recognizing phospho-tau, under conditions being suitable for producing an antigen-antibody complex;
detecting the immunological binding of said antibody to said sample.
In another embodiment, phospho-tau can be quantified by a sandwich ELISA comprising the following steps:
obtaining a sample from the patient;
bringing said sample into contact with an antibody (primary antibody or capturing antibody) recognizing phospho-tau, under conditions being suitable for producing an antigen-antibody complex;
bringing said sample into contact with a monoclonal antibody (secondary antibody or detector antibody) specifically recognizing phospho-tau, under conditions being suitable for producing an antigen-antibody complex;
bringing the antigen-antibody complex into contact with a marker either for specific tagging or coupling with said secondary antibody, with said marker being any possible marker known to the person skilled in the art;
possibly also, for standardization purposes, bringing the antibodies in contact with a purified phospho-tau protein or phospho-peptide reactive with both antibodies.
Advantageously, the secondary antibody itself carries a marker or a group for direct or indirect coupling with a marker.
The expression xe2x80x98recognizingxe2x80x99, xe2x80x98reacting withxe2x80x99, xe2x80x98immunological bindingxe2x80x99 or xe2x80x98producing an antigen-antibody complexxe2x80x99 as used in the present invention is to be interpreted that binding between the antigen and antibody occurs under all conditions that respect the immunological properties of the antibody and the antigen.
The expression xe2x80x98specifically recognizingxe2x80x99 as used in the present invention is to be interpreted that said antibody is capable of forming an immunological complex with phospho-tau but not with human normal tau.
Any monoclonal antibody that specifically recognizes phospho-tau can be used in said method for the quantification of phospho-tau. Monoclonal antibodies for use in the quantification of phospho-tau include AT8 (WO 93/08302), AT180 and AT270 (WO 95/17429) and AT100 (WO 96/04309). Other antibodies known in the art that specifically recognize phospho-tau can be used as well.
Also fragments derived from these monoclonal antibodies such as Fab, F(ab)xe2x80x22, ssFv (xe2x80x98single chain variable fragmentxe2x80x99) and other antibody-like constructs that retain the variable region of the antibody, providing they have retained the original binding properties, can be used in a method of the present invention. Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses. Also mini-antibodies and multivalent antibodies such as diabodies, triabodies, tetravalent antibodies and peptabodies can be used in a method of the invention. The preparation and use of these fragments and multivalent antibodies has been described extensively in International Patent Application WO 98/29442.
The monoclonal antibodies used in a method of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains. Alternatively the monoclonal antibodies used in a method of the invention may be human monoclonal antibodies. The term xe2x80x98humanized antibodyxe2x80x99 means that at least a portion of the framework regions of an immunoglobulin is derived from human immunoglobulin sequences.
The antibodies used in a method of the present invention may be labeled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
The method for the in vivo detection of the level of phospho-tau in an individual comprises the steps of determining the level of phospho-tau in said individual and comparing it with a previously defined phospho-tau level range characteristic for AD, DLB, PD without dementia, MSA or PSP. In an embodiment of the invention, phospho-tau can be quantified by in vivo imaging. Phospho-tau can be quantified in situ by non-invasive methods including but not limited to brain imaging methods described by Arbit et al. (1995), Tamada et al. (1995), Wakabayashi et al. (1995), Huang et al. (1996), Sandrock et al. (1996), Mariani et al. (1997). These in vivo imaging methods may allow the localization and quantification of phospho-tau, for example, by use of labeled antibodies specifically recognizing phospho-tau.
The present invention thus relates to the use of phospho-tau as a neurological marker for the differential diagnosis of an individual suffering from AD versus an individual suffering another neurological disease. The present invention specifically relates to the use of phospho-tau as a neurological marker for the differential diagnosis of an individual suffering from AD versus an individual suffering from DLB, versus an individual suffering from PD without dementia, versus an individual suffering from MSA and/or versus an individual suffering from PSP.
The present invention also relates to the use of phospho-tau as a neurological marker for the manufacture of a diagnostic kit for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from another neurological disease. The present invention specifically relates to the use of phospho-tau as a neurological marker for the manufacture of a diagnostic kit for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from DLB, versus an individual suffering from PD without dementia, versus an individual suffering from MSA and/or versus an individual suffering from PSP.
The present invention also relates to the use of an antibody that specifically recognizes phospho-tau for the manufacture of a diagnostic kit for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from another neurological disease. The present invention specifically relates to the use of an antibody that specifically recognizes phospho-tau for the manufacture of a diagnostic kit for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from DLB, versus an individual suffering from PD without dementia, versus an individual suffering from MSA and/or versus an individual suffering from PSP.
In accordance, the present invention also relates to a diagnostic kit for use in the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from another neurological disease, comprising an antibody that specifically recognizes phopho-tau. The present invention specifically relates to a diagnostic kit for use in the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from DLB, versus an individual suffering from PD without dementia, versus an individual suffering from MSA and/or versus an individual suffering from PSP, comprising an antibody that specifically recognizes phopho-tau.
A possible kit for carrying out the method of the invention is based on an immunoassay and comprises:
an antibody (primary antibody) which forms an immunological complex with an epitope of phospho-tau;
a monoclonal antibody (secondary antibody) which specifically recognizes phospho-tau;
a marker either for specific tagging or coupling with said secondary antibody;
appropriate buffer solutions for carrying out the immunological reaction between the primary antibody and the test sample, between the secondary antibody and the test sample and/or between the bound secondary antibody and the marker;
possibly, a purified phospho-tau protein or a phospho-peptide for standarization purposes.
The present invention further relates to the use of a diagnostic kit as described above for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from another neurological disease. The present invention specifically relates to the use of a diagnostic kit as described above for the differential diagnosis of an individual suffering from Alzheimer""s disease versus an individual suffering from DLB, versus an individual suffering from PD without dementia, versus an individual suffering from MSA and/or versus an individual suffering from PSP.
The invention also relates to a method to screen or monitor the effect on an individual of compounds which prevent or treat Alzheimer""s disease, dementia with Lewy bodies, Parkinson""s disease without dementia, multi-system atrophy and/or progressive supranuclear paly.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, and variations such as xe2x80x9ccomprisesxe2x80x9d and xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or step or group of stated integers or steps but not to the exclusion of any other integer or step or group of integers or steps.
The present invention will now be illustrated by reference to the following examples that set forth particularly advantageous embodiments. However, it should be noted that these examples are illustrative and can not be construed as to restrict the invention in any way.